A microbial biofilm is a community of microbial cells embedded in an extracellular matrix of polymeric substances and adherent to a biological or a non-biotic surface. A range of microorganisms (bacteria, fungi, and/or protozoa, with associated bacteriophages and other viruses) can be found in these biofilms. Biofilms are ubiquitous in nature, are commonly found in a wide range of environments. Biofilms are being increasingly recognised by the scientific and medical community as being implicated in many infections, and especially their contribution to the recalcitrance of infection treatment.
Biofilms are etiologic agents for a number of disease states in mammals and are involved in 80% of infections in humans. Examples include skin and wound infections, middle-ear infections, gastrointestinal tract infections, peritoneal membrane infections, urogenital tract infections, oral soft tissue infections, formation of dental plaque, eye infections (including contact lens contamination), endocarditis, infections in cystic fibrosis, and infections of indwelling medical devices such as joint prostheses, dental implants, catheters and cardiac implants.
Planktonic microbes (i.e., microorganisms suspended or growing in a liquid medium) are typically used as models for antimicrobial susceptibility research as described by the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Microbes in biofilms are significantly more resistant to antimicrobial treatment than their planktonic counterparts. However, there is no standardized method for the study of antibiotic susceptibility of biofilm microbes.
Biofilm formation is not limited solely to the ability of microbes to attach to a surface. Microbes growing in a biofilm are able to interact more between each other than with the actual physical substratum on which the biofilm initially developed. For example, this phenomenon favours conjugative gene transfer, which occurs at a greater rate between cells in biofilms than between planktonic cells. This represents an increased opportunity for horizontal gene transfer between bacteria, and is important because this can facilitate the transfer of antibiotic resistance or virulence determinant genes from resistant to susceptible microbes. Bacteria can communicate with one another by a system known as quorum sensing, through which signalling molecules are released into the environment and their concentration can be detected by the surrounding microbes. Quorum sensing enables bacteria to co-ordinate their behaviour, thus enhancing their ability to survive. Responses to quorum sensing include adaptation to availability of nutrients, defense against other microorganisms which may compete for the same nutrients and the avoidance of toxic compounds potentially dangerous for the bacteria. It is very important for pathogenic bacteria during infection of a host (e.g. humans, other animals or plants) to co-ordinate their virulence in order to escape the immune response of the host in order to be able to establish a successful infection.
Biofilm formation plays a key role in many infectious diseases, such as cystic fibrosis and periodontitis, in bloodstream and urinary tract infections and as a consequence of the presence of indwelling medical devices. The suggested mechanisms by which biofilm-associated microorganisms elicit diseases in their host include the following: (i) delayed penetration of the antimicrobial agent through the biofilm matrix, (ii) detachment of cells or cell aggregates from indwelling medical device biofilms, (iii) production of endotoxins, (iv) resistance to the host immune system, (v) provision of a niche for the generation of resistant organisms through horizontal gene transfer of antimicrobial resistance &/or virulence determinant genes, and (vi) altered growth rate (.i.e. metabolic dormancy) (Donlan and Costerton, Clin Microbiol Rev 15: 167-193, 2002; Parsek and Singh, Annu Rev Microbiol 57: 677-701, 2003; Costerton J W, Resistance of biofilms to stress. In ‘The biofilm primer’. (Springer Berlin Heidelberg). pp. 56-64.2007).
Recent experimental evidence has indicated the existence within biofilms of a small sub-population of specialized non-metabolising persister cells (dormant cells). It is thought that these cells may be responsible for the high resistance/tolerance of biofilm to antimicrobial agents. Multi-drug-tolerant persister cells are present in both planktonic and biofilm populations and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to this sub-population. The protection offered by the polymeric matrix allows persister cells to evade elimination and serve as a source for re-population. There is evidence that persisters may be largely responsible for the multi-drug tolerance of microbial biofilms (LaFleur et al., Antimicrob Agents Chemother. 50: 3839-46, 2006; Lewis, Nature Reviews Microbiology 5, 48-56 2007).
There remains a need for better therapies for preventing biofilm formation and treating conditions associated with microbial biofilms.
Table 1: Summary of the activity of the tested antimicrobial agents against the Gram-negative P. aeruginosa strains and the Gram-positive Staphylococcus spp.
Table 2: Summary of the activity of the tested antimicrobial agents against S. epidermidis, S. aureus, and P. aeruginosa. 
The present invention relates to a product comprising at least two antibiofilm agents wherein at least one of the antibiofilm agents is an antimicrobial peptide. The second antibiofilm agent is generally a dispersant or an anti-adhesive agent. There is also provided the use of the product in the treatment of a microbial infection.
TABLE 1Exp#1-2MIC (μg/m1) at pH 7S. epidermidisS. aureusS. aureusP. aeruginosaP. aeruginosaP. aeruginosaNPSequenceATCC12228ATCC25923DSMZ11729DSMZ1128DSMZ1299ATCCBAA-47NP432RRRFRFFFRFRRR<7.831.2562.562.515.615.6 NP438HHHFRFFFRFRRR<7.8>500>500>500500>500 NP441HHPRRKPRRPKRHH>500>500>500>500>500>500 NP445KKFPWRLRLRYGRR<7.850050062.531.2531.25 NP449KKPRRKPRRPKRKK-31.25250125250125250cyst NP451HHPRRKPRRPKRHH-125500500>500>500>500cyst NP457RRRRR-cyst31.25125125>500>500250 NP458RRRRRHH-cyst31.2525025025012562.5
TABLE 2MBC (μg/ml) following MIC at pH 7Exp#3P. aeruginosaS. epidermidisS. aureusS. aureusP. aeruginosaP. aeruginosaP. aeruginosaP. aeruginosaNPDSMZ1299ATCC12228ATCC25923DSMZ11729DSMZ1128DSMZ1299ATCCBAA-47DSMZ1299NP432 16  250  500250 (2) 32 (2)  250NP438125>500>500>500>500>500NP441>500>500  >500>500>500>500>500>500NP445  62.5>500>500  250  125  250NP449125>500  250500 (2)250 (2)>500NP451>500125>500>500>500>500>500>500NP457>500125 (2)    62.5125 (2)>500>500>500>500NP458500  125  250>500>500>500Exp#3Exp#4MIC (μg/ml) pH 5.5,Exp#4MBC (μg/ml) pH 5.5,MIC (μg/ml) pH 5.5320 mM NaClMBC (μg/ml) at pH 5.5320 mM NaClS. aureusP. aeruginosaS. aureusP. aeruginosaS. aureusP. aeruginosaS. aureusP. aeruginosaNP11729ATCCBAA-4711729ATCCBAA-4711729ATCCBAA-4711729ATCCBAA-47NP432>500  125>500  125>500  125>500>500NP438>500  125>500    62.5>500>500>500>500NP441>500>500>500>500>500>500>500>500NP445>500>500>500>500>500>500>500>500NP449>500>500>500>500>500>500>500>500NP451>500>500>500>500>500  250>500>500NP457>500>500>500>500>500>500>500>500NP458>500>500>500>500>500>500>500>500